The in vivo and in vitro metabolism of cyclophosphamide (CP) is being investigated in an effort to evaluate the role of different metabolites in toxicity and chemotherapeutic activity of CP. Both 3H-chloroethyl CP and 14(C-4)-CP are being used to isolate and identify the metabolites that interact with macromolecules such as proteins and nucleic acids. Acrolein binds to microsomal proteins and the consequence of this binding is the denaturation of cytochrome P-450. CP also causes depression of mixed function oxygenase (cytochrome P-450) in vivo and this is dependent upon the age and sex of the animal, and is also dose-, and time-dependent. However, this effect of CP can blocked by N-acetylcysteine given concomitantly with CP. Cysteine, although comparatively weaker than N-acetyl cysteine, also affords some protection against CP-induced in vivo depression of cytochrome P-450 and hematuria; however, at the same time, it spares the chemotherapeutic activity of CP against Walker 256 Carcinoma in rats. The nature of the CP metabolites interacting with macromolecules, the nature of these interactions and the role of glutathione in the metabolism-dependent bioligical effect of CP are under investigation.